Abstract
Mixed infection of tissue culture cells is the primary means of studying genetic and nongenetic interactions between viral mutants. The purpose of the mixed infection is to place two different viral genomes into the same cell, where interactions between the genomes and their encoded gene products can take place. In some cases, interactions are observed in the context of the mixed infected cell, but generally the yield of progeny virus from the mixed infected cells is assayed to reveal the result of the interactions. Here, methods for mixed infections between conditional-lethal mutants of the temperature-sensitive (ts) phenotype will be presented and considered. (Mutants with ts phenotype are designated according to the following nomenclature: tsA [778], in which ts indicates the phenotype, A indicates the mutant belongs to recombination group A, and [778] indicates the number of the specific mutant in group A.) Temperature-sensitive mutants are particularly useful for genetic studies, because they can be propagated at the permissive temperature (PT; 31°C in the case of rotavirus [RV] ts mutants), but the mutant phenotype is expressed at the nonpermissive temperature (NPT; 39°C in the case of RV ts mutants). The conditional-lethality of the ts mutations allows the use of selective conditions in the analysis of the progeny of the mixed infections, so that the results of genetic and nongenetic interactions can be easily revealed.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fields, B. N. (1996) Reoviridae, in Field’s Virology, 3rd ed., (Fields, B. N., Knipe, D. M., Howley, P. M., et al., eds.), Lippincott-Raven, Philadelphia, pp. 1553–1555.
Ramig, R. F. and Ward, R. L. (1991) Genomic segment reassortment in rotaviruses and other reoviridae. Adv.Virus Res. 39, 163–207.
Ramig, R. F. (1997) Genetics of the rotaviruses. Annu. Rev. Microbiol. 51, 225–255.
Greenberg, H. B., Kalica, A. R., Wyatt, R. W., Jones, R. W., Kapikian, A. Z., and Chanock, R. M. (1981) Rescue of noncultivable human rotavirus by gene reassortment during mixed infection with ts mutants of a cultivable bovine rotavirus. Proc. Natl. Acad. Sci. USA 78, 420–424.
Ramig, R. F. (1982) Isolation and genetic characterization of temperature-sensitive mutants of simian rotavirus SA11. Virology 120, 93–105.
Ramig, R. F. (1983) Isolation and genetic characterization of temperature-sensitive mutants that define five additional recombination groups in simian rotavirus SA11. Virology 130, 464–473.
Ramig, R. F. (1983) Factors that affect genetic interaction during mixed infections with temperature-sensitive mutants of simian rotavirus SA11. Virology 127, 91–99.
Faulker-Valle, G. P., Clayton, A. V., and McCrae, M. A. (1982) Molecular biology of rotaviruses. III. Isolation and characterization of temperature-sensitive mutants of bovine rotavirus. J. Virol. 42, 669–677.
Chen, D. and Ramig, R. F. (1994) Construction and characterization of rotavirus reassortants. Methods Mol. Genet. 4, 183–194.
Rubin, D. H. and Fields, B. N. (1980) Molecular basis of reovirus virulence: Role of the M2 gene. J. Exp. Med. 152, 853–868.
Ramig, R. F., Garrison, C., Chen, D., and Bell-Robinson, D. (1989) Analysis of reassortment and superinfection during mixed infection of Vero cells with bluetongue virus serotypes 10 and 17. J. Gen. Virol. 70, 2595–2603.
Graham, A., Kudesia, G., Allen, A. M., and Desselberger, U. (1987) Reassortment of human rotavirus possessing genome rearrangements with bovine rotavirus: nonrandomness and evidence for host cell selection. J. Gen. Virol. 68, 115–122.
Chen, D., Burns, J. W., Estes, M. K., and Ramig, R. F. (1989) The phenotypes of rotavirus reassortants depend upon the recipient background. Proc. Natl. Acad. Sci. USA 86, 3743–3747.
Chen, D., Estes, M. K., and Ramig, R. F. (1992) Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a crossreactive, neutralizing VP4-specific epitope. J. Virol. 66, 432–439.
Gombold, J. L. and Ramig, R. F. (1987) Assignment of simian rotavirus SA11 temperature-sensitive mutant groups A, C, F, and G to genome segments. Virology 161, 463–473.
Gombold, J. L., Estes, M. K., and Ramig, R. F. (1985) Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments. Virology 143, 309–320.
Ramig, R. F. (1990) Superinfecting rotaviruses are not excluded from genetic interactions during asynchronous mixed infections in vitro. Virology 176, 308–310.
Tauscher, G. I. and Desselberger, U. (1997) Viral determinants of rotavirus pathogenicity in pigs: Production of reassortants by asynchronous coinfection. J. Virol. 71, 853–857.
Matsuno, S., Inouye, S., and Kono, R. (1977) Plaque assay of neonatal calf diarrhea virus and the neutralizing antibody in human sera. J. Clin. Microbiol. 5, 1–4.
Smith, E. M., Estes, M. K., Graham, D. Y., and Gerba, C. P. (1979) A plaque assay for the simian rotavirus SA11. J. Gen. Virol. 43, 513–519.
Chakraborty, P. R., Ahmed, R., and Fields, B. N. (1979) Genetics of reovirus: the relationship of interference to complementation and reassortment of temperature-sensitive mutants at nonpermissive temperature. Virology 94, 119–127.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Ramig, R.F. (2000). Mixed Infections with Rotaviruses. In: Gray, J., Desselberger, U. (eds) Rotaviruses. Methods in Molecular Medicine™, vol 34. Humana Press. https://doi.org/10.1385/1-59259-078-0:79
Download citation
DOI: https://doi.org/10.1385/1-59259-078-0:79
Publisher Name: Humana Press
Print ISBN: 978-0-89603-736-6
Online ISBN: 978-1-59259-078-0
eBook Packages: Springer Protocols